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BB1702 Biochemistry: Structure and Function > Separation techniques > Flashcards

Flashcards in Separation techniques Deck (16)
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1

What's the first step in determining a protein sequence?

Purification

2

What can proteins be seperated based on?

  • solubility
  • size
  • charge
  • binding ability

3

How are proteins isolated from an assay?

By centrifuge

4

Describe dialysis

A seperation technique that seperates molecules based on size using a semipermeable membrane e.g. cellulose

5

Describe gel-filtration chromatography

A seperation technique that seperates based on size. The sample is applied to the top of the column consisting of porous beads. The large molecules flow more rapidly through the column.

6

Describe ion-exchange chromatography

A separation technique that separates based on net-charges. 

If a protein has a net positive charge (at pH7), it will bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not.

The bound protein can then be eluted.

7

Describe affinity chromatography

A separation technique that separates based on the chemical groups present.
This technique takes advantages of the high affinity of many proteins for specific chemical groups.

Good for isolating transcription factors.

8

Describe high-pressure liquid (HPLC) chromatography

  • Is an enhanced version of column separation
  • Column materials more refined
  • Increased resolution and flow rate

9

What is the next stage in separation after the purification of proteins?

Separation of the purified proteins by gel electrophoresis

10

The principle of ___ _____________ is that a molecule with a net charge will move in an electric field. The velocity of _________ (v) of a protein in an electric field depends on the ________ _______ _________ (E), the ___ _______ of the protein (z), and the __________ ___________ (f).

The principle of gel electrophoresis is that a molecule with a net charge will move in an electric field. The velocity of migration (v) of a protein in an electric field depends on the electric field strength (E), the net charge of the protein (z), and the frictional coefficient (f).

11

V = Ez/f

Velocity of migration of a protein = (electric field strength X net charge of protein) / frictional coefficient

12

What does the frictional coefficient of a protein depend upon?

The mass and shape of the migrating molecule, and the viscocity of the medium

13

Describe polyacrylamide gel electrophoresis  (PAGE)

  • Electrophoresis is performed on a thin, vertical slab of polyacrylamide gel
  • The gel acts as a molecular sieve
  • The gel is inert
  • All molecules are forced to move through the same matrix
  • Smaller molecules move faster

14

Describe SDS-PAGE electrophoresis

  • The proteins are first dissolved in SDS to disrupt non-covalent interactions
  • Small proteins move rapidly through gel, large ones stay near top
  • mobility of proteins is linearly proportional to the log of their mass

15

Describe isoelectric focusing

A separation technique based on isoelectric points. Each protein moves until it reaches a position in the gel at which the pH = pI of the protein.

The gel gradient is formed by subjecting a mixture of polyampholytes (small multicharged polymers) which have many pI values to electrophoresis.

16

Describe two-dimensional electrophoresis

Isoelectric focusing is combined with SDS-PAGE to obtain very high resolution separations.

The proteins are subjected to IF and then horizontal SDS-PAGE.